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The human CRISPR activation library (Calabrese P65-HSF) is in backbone pXPR_502 (P65 HSF). This Calabrese library activates over 18,000 human genes and is used for genome-wide activation screening.

Each gene activated by this library is targeted by 3-6 gRNAs. These gRNAs are split among the two pools: Set A (Cat# 92379) contains gRNAs 1-3, Set B (Cat# 92380) contains gRNAs 4-6. Each pool has a unique set of gRNAs, but both pools are designed to target the same genes. The slight discrepancy in number of genes targeted between Set A and Set B is because not all genes had 6 gRNAs.

Depositor comments: The modified sgRNA tracr has two MS2 loops and two PP7 loops, although only the PP7 loops are used in this library. This library uses two vectors instead of 3, as the PP7-P65-HSF is on same vector as sgRNA itself. It was found that MS2-P65-HST (hygroR) vector gave poor titer. The 6 guides per gene were designed first based on location relative to TSS, then sequence preferences.

Ordering

Human CRISPRa sgRNA library Calabrese (P65 HSF), Set A & B (entire library)
A dCas9 activation plasmid is NOT included with this item and will have to be ordered separately. Can be used in conjunction with lenti dCAS-VP64_Blast (Addgene #61425).

Human CRISPRa sgRNA library Calabrese (P65 HSF), Set AA dCas9 activation plasmid is NOT included with this item and will have to be ordered separately. Can be used in conjunction with lenti dCAS-VP64_Blast (Addgene #61425).

Human CRISPRa sgRNA library Calabrese (P65-HSF), Set BA dCas9 activation plasmid is NOT included with this item and will have to be ordered separately. Can be used in conjunction with lenti dCAS-VP64_Blast (Addgene #61425).

Viral Production & Use

Biosafety

Requestor is responsible for compliance with
their institution's biosafety regulations.
Lentivirus is generally considered BSL-2. AAV is
generally considered BSL-1, but may require
BSL-2 handling depending on the insert.
Biosafety Guide

Viral Quality Control

Pooled Library Representation:

To confirm library representation and distribution, we perform next-generation sequencing on the purified plasmid DNA pool that was used to generate lentivirus.

Titering Method:

Colony formation assay: A549 cells were transduced with serial dilutions of 92379-LV and treated with puromycin. Puromycin-resistant colonies were expanded for approximately 2 weeks, stained with crystal violet, and counted.

PCR confirmation of insert:

PCR was carried out with primers targeting the U6 and PGK promoters. The PCR product was visualized on an agarose gel for size confirmation.

Addgene Comments

Shipment specifications:

Pooled libraries are shipped on dry ice and aliquoted among several tubes, for a total of at least 1.15x10⁸ transducing units. Each viral service request also includes virus associated DNA, which is a sample of the purified plasmid DNA pool that was used to generate lentivirus.

How to use this virus:

We recommend using one aliquot of virus to determine optimal infection conditions before performing screening-scale infections. Detailed methods for determining optimal infection conditions, screening, and validating hits are described in a related publication. Before using this virus, Addgene strongly recommends reading this related publication from the same lab (Doench et al., 2016).

A brief and partial description of how to use this virus:

Start by optimizing the infection conditions in your cell line in order to achieve 30-50% infection efficiency, corresponding to a multiplicity of infection (MOI) of ~0.5–1. Infect 2.5×106 suspension (or 3×106 adherent) cells in each well of a 12-well dish at different MOIs. Determine which condition yields a 30-50% infection efficiency, and note the infection efficiency for this condition.

The Calabrese CRISPR activation pooled library (set A) has 56,762 gRNAs. To achieve the recommended representation of ~400 cells per sgRNA, a total of ~2.3×107 infected cells is needed. Calculate the number of cells on which to perform the infection by considering the infection efficiency noted in the optimized condition. For example, if the infection efficiency is 50%, perform the infection on 4.5×107 cells to ultimately achieve 2.3×107 infected cells.

Perform the screening-scale infection with the pre-determined MOI in the same 12-well format as the optimized condition. Briefly, infect 2.5×106 suspension (or 3×106 adherent) cells in each well of a 12-well dish. Scale up the number of wells to achieve the desired total number of cells.

Based on a screening MOI of 0.5-1 and the total number of cells infected, Addgene estimates that 11-36 mL of virus will be needed to perform the screen. This corresponds to 2.3×107-7.6×107 TUs. An additional 1 mL of virus will be needed to perform the optimization. Based on the infection efficiency observed in each cell line, these estimates are subject to variation. Not every cell type is infectable under these conditions, and Addgene recommends that infectability of cells be determined empirically.

How to use the virus associated DNA:

Distribution of this pooled lentiviral library includes virus associated DNA, which is a sample of the purified plasmid DNA pool that was used to generate lentivirus.

This purified plasmid DNA pool can be sequenced and used as the starting plasmid DNA (pDNA) pool reference when performing screen analysis. As described in a related publication (Doench et al., 2016), perform screen analysis by determining the log2-fold-change of each single-guide RNA relative to the pDNA pool. The pDNA has been shown to serve as a good surrogate for an early time point and is more cost effective when performing many screens (Shalem et al., 2014).

Viral Production & Use

Biosafety

Requestor is responsible for compliance with
their institution's biosafety regulations.
Lentivirus is generally considered BSL-2. AAV is
generally considered BSL-1, but may require
BSL-2 handling depending on the insert.
Biosafety Guide

Viral Quality Control

Pooled Library Representation:

To confirm library representation and distribution, we perform next-generation sequencing on the purified plasmid DNA pool that was used to generate lentivirus.

Titering Method:

Colony formation assay: A549 cells were transduced with serial dilutions of 92380-LV and treated with puromycin. Puromycin-resistant colonies were expanded for approximately 2 weeks, stained with crystal violet, and counted.

PCR confirmation of insert:

PCR was carried out with primers targeting the U6 and PGK promoters. The PCR product was visualized on an agarose gel for size confirmation.

Addgene Comments

Shipment specifications:

Pooled libraries are shipped on dry ice and aliquoted among several tubes, for a total of at least 1.15x10⁸ transducing units. Each viral service request also includes virus associated DNA, which is a sample of the purified plasmid DNA pool that was used to generate lentivirus.

These pooled libraries were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the pooled libraries were described, and include Addgene in the Materials and Methods of your future publications.

Example for your Materials & Methods section:

Human Calabrese CRISPR activation pooled library set A was a gift from David Root and John Doench (Addgene #92379). orHuman Calabrese CRISPR activation pooled library set B was a gift from David Root and John Doench (Addgene #92380).